Intra- and inter-molecular regulation by intrinsically-disordered regions governs PUF protein RNA binding

PUF proteins are characterized by globular RNA-binding domains. They also interact with partner proteins that modulate their RNA-binding activities. Caenorhabditis elegans PUF protein fem-3 binding factor-2 (FBF-2) partners with intrinsically disordered Lateral Signaling Target-1 (LST-1) to regulate target mRNAs in germline stem cells. Here, we report that an intrinsically disordered region (IDR) at the C-terminus of FBF-2 autoinhibits its RNA-binding affinity by increasing the off rate for RNA binding. Moreover, the FBF-2 C-terminal region interacts with its globular RNA-binding domain at the same site where LST-1 binds. This intramolecular interaction restrains an electronegative cluster of amino acid residues near the 5′ end of the bound RNA to inhibit RNA binding. LST-1 binding in place of the FBF-2 C-terminus therefore releases autoinhibition and increases RNA-binding affinity. This regulatory mechanism, driven by IDRs, provides a biochemical and biophysical explanation for the interdependence of FBF-2 and LST-1 in germline stem cell self-renewal.

For all statistical analyses, confirm that the following items are present in in the figure legend, table legend, main text, or or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as as a discrete number and unit of of measurement A statement on on whether measurements were taken from distinct samples or or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of of all covariates tested
A description of of any assumptions or or corrections, such as as tests of of normality and adjustment for multiple comparisons A full description of of the statistical parameters including central tendency (e.g.means) or or other basic estimates (e.g.regression coefficient) AND variation (e.g. standard deviation) or or associated estimates of of uncertainty (e.g.confidence intervals) For null hypothesis testing, the test statistic (e.g.F, t, r) with confidence intervals, effect sizes, degrees of of freedom and P value noted Give P values as exact values whenever suitable.
For Bayesian analysis, information on on the choice of of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of of the appropriate level for tests and full reporting of of outcomes Estimates of of effect sizes (e.g.Cohen's d, Pearson's r), ), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.All manuscripts must include a data availability statement.This statement should provide the following information, where applicable:

Software and code
-Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Research involving human participants, their data, or biological material Policy information about studies with human participants or human data.See also policy information about sex, gender (identity/presentation), and sexual orientation and race, ethnicity and racism.

Reporting on sex and gender
Reporting on race, ethnicity, or other socially relevant groupings

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research.If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Blinding
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
Atomic coordinates and structure factors for the reported crystal structure have been deposited with the Protein Data Bank under accession number 8SJ7.PDB 3V74 was used as a model for molecular replacement.All other data supporting the findings of this study are available within the paper and its Supplementary Information.Source data are provided with this paper.
Not applicable, no human participants, data, or biological material were used.

Not applicable
Not applicable

Not applicable
For in vitro assays: EMSAs were conducted at least in triplicate and values are reported as Kd ± SEM.This is a customary sample size, which provides the power to detect statistically significant differences, if present.ITC was conducted in duplicate due to the high amount of sample required for an individual replicate, and values for both experiments, which are similar, are reported.SPR was conducted in duplicate at 5 protein concentrations and values are reported as mean ± SD.Duplicate SPR experiments were performed at 5 protein concentrations, representing a sample size of 10.
No data were excluded.
Technical replicates for EMSAs, ITC, and SPR experiments produced similar results.
This section is not applicable to our in vitro studies.
We did not blind the data processing from the data collection.Dr. Qiu performed experiments and also processed the data.
Policy information about availability of of computer code Data collection Data analysis nature portfolio | reporting summary April 2023 Data Policy information about availability of data